Ligand-induced activation and G protein coupling of prostaglandin F2α receptor

Prostaglandin F2α (PGF2α), an endogenous arachidonic acid metabolite, regulates diverse physiological functions in many tissues and cell types through binding and activation of a G-protein-coupled receptor (GPCR), the PGF2α receptor (FP), which also is the primary therapeutic target for glaucoma and several other diseases. Here, we report cryo-electron microscopy (cryo-EM) structures of the human FP bound to endogenous ligand PGF2α and anti-glaucoma drugs LTPA and TFPA at global resolutions of 2.67 Å, 2.78 Å, and 3.14 Å. These structures reveal distinct features of FP within the lipid receptor family in terms of ligand binding selectivity, its receptor activation, and G protein coupling mechanisms, including activation in the absence of canonical PIF and ERY motifs and Gq coupling through direct interactions with receptor transmembrane helix 1 and intracellular loop 1. Together with mutagenesis and functional studies, our structures reveal mechanisms of ligand recognition, receptor activation, and G protein coupling by FP, which could facilitate rational design of FP-targeting drugs.

Prostanoids are a class of oxygenated arachidonic acid metabolites that include prostaglandin F 2α (PGF 2α ), prostaglandin D 2 (PGD 2 ), prostaglandin E 2 (PGE 2 ), thromboxane A 2 (TXA 2 ), and prostacyclin (PGI 2 ). They provoke diverse biological actions in many tissues and cell types through direct interactions with nine lipid G-protein-coupled receptors (GPCRs), prostaglandin F 2α receptor (FP), prostaglandin D 2 receptors (DP1-2), prostaglandin E 2 receptors (EP1-4), thromboxane receptor (TP) and prostacyclin receptor (IP1) 1 , which comprise a subfamily of class A GPCRs. FP is encoded in humans by the PTGFR gene 2 . Stimulated by PGF 2α , FP plays a pivotal role in regulating inflammation, allergic responses, intraocular pressure, and blood pressure, making it a valuable target for therapeutic discovery and development 1,[3][4][5] . FP is highly expressed in uterine myometrium, eye, smooth muscle, skin, and ovarian 6,7 . Upon agonist stimulation, FP is predominantly coupled to the G q subtype of G proteins, which activation leads to subsequent PKC activation and a transient calcium signaling in response to the formation of inositol triphosphate 8,9 . In addition to G q , FP activation also induces activation of small G protein Rho via G 12 /G 13 10 and activation of Raf/MEK/MAP kinase pathway through Gi 11 .
Given the diverse functions of FP, it has been targeted for therapeutic development. PGF 2α , the endogenous FP activator, entered a clinical trial for glaucoma treatment 12 . However, its clinical application was limited by intolerable side effects, possibly caused by its low selectivity for FP receptor 12 . Since then, selective FP agonists have attracted extensive attention and have been developed for the treatment of glaucoma 5 , scalp alopecia 13 , and vitiligo 14 . From 1996 to 2012, several FP-selective prostaglandin analogs (PGAs) were approved by the United States Food and Drug Administration (FDA) for glaucoma treatment. As a prodrug of a selective FP receptor agonist, latanoprost (LTP) was approved to treat glaucoma for the first time in 1996. It has also been used to treat scalp alopecia 15 and vitiligo 14 in recent years. Latanoprost acid (LTPA), 17-phenyl-13,14-dihydro PGF 2α , is an active metabolic form of LTP. Another fluorinated PGA prodrug, tafluprost (TFP), was first approved for the treatment of glaucoma in 2012 16 . Tafluprost acid (TFPA), 15-deoxy-15,15-difluoro-16-phenoxy PGF 2α , is the active metabolic form of TFP. Due to their high efficacy, these PGAs have been the first-line drug in clinics for the treatment of glaucoma. However, through post-marketing surveillance, 5-20% of patients suffered side effects such as conjunctival congestion and headache, including intolerance responses toward these PGAs [17][18][19] . Improving the selectivity of these drugs to the FP receptor and elucidating the molecular mechanisms underlying the functional selectivity of individual prostanoid receptor family members are highly important and clinically relevant.
Extensive efforts have been made to clarify how the binding of endogenous and synthetic ligands with various pharmacological profiles regulate FP's downstream signaling [8][9][10][11] . However, the molecular details defining the binding modes of ligands remain largely unknown, which is partly attributed to the scarcity of the structural information on ligands bound FP complex. Understanding the mechanism of prostaglandin-FP signaling and identifying differences in the ligand selectivity of prostaglandin receptors may assist in the development of selective drugs with improved safety.
Here we present three cryo-EM structures of G protein-coupled FP in complex with its endogenous ligand PGF 2α and with two synthetic agonists, LTPA and TFPA, at global resolutions of 2.67, 2.78, and 3.14 Å. Combined with functional characterizations of mutated receptors, these structures reveal conserved and divergent mechanisms of ligand binding, receptor activation, and G protein coupling by FP.

Cryo-EM analysis and overall structure
To facilitate the expression of FP complexes, we introduced a BRIL tag to the N-terminus of the wild-type (WT) receptor 20,21 . A Gα q chimera was engineered based on the mini-Gα s scaffold with an N-terminal replacement of corresponding sequences of Gα i1 to facilitate the binding of scFv16 [22][23][24] . Hereinafter, Gα q reference to Gα q chimera. The FP-G q complex was further stabilized by the NanoBiT strategy 25 . Incubation of PGF 2α /LTPA/TFPA with membranes from cells co-expressing receptors and heterotrimer G q proteins in the presence of scFv16 and Nb35 enables efficient assembly of the PGF 2α /LTPA/TFPA -FP-G q complexes, which produces highly homogenous complex samples for structural studies 26 (Supplementary Figs. 1-3, Table 1). The structures of the FP-G q -scFv16-Nb35 complexes with PGF 2α , LTPA, and TFPA were determined by cryo-EM to the resolutions of 2.67, 2.78, and 3.14 Å (Fig. 1a, Supplementary Figs. 1-4). The high-quality density map allowed unambiguous model building for the receptor structure containing residues 29-323, except for two invisible residues in the intracellular loop 3 (ICL3) (residues 238 and 239). The density maps are also clear for three agonists, most residues of the G q heterotrimer, scFv16, and Nb35 (Fig. 1b, c Supplementary Fig. 4).
The overall structure of the active FP receptor is highly similar to those of active EP2 (PDB code: 7CX2) and EP4 (PDB code: 7D7M), with root mean square deviation (RMSD) values of 1.27 and 1.23 Å, respectively. FP folds into a canonical seven-transmembrane helical domain (TMD). All three extracellular loops (ECLs 1-3) were well defined, where ECL2 forms a β-hairpin loop, which is stabilized by the highly conserved disulfide bond between C186 ECL2 and C108 3.25 . The β-hairpin ECL2 of FP resembles those of EP2 and EP4, and tightly caps the extracellular region (Fig. 1d). We superpose the TMD structures of PGF 2α -bound FP with PGE 2 -bound EP2 and EP4 to compare their overall receptor conformations and ligand-binding pockets. A notable structural difference occurs in the H8 of these receptors. The H8 of FP is almost perpendicular to that of EP2, with a rotation of 82.3°, and is closer to the cell membrane compared with the H8 of EP4. (Fig. 1e). In addition, although these ligands share a similar chemical scaffold and relatively conserved binding pocket in these three receptors, PGF 2α displays a distinct binding pose from PGE 2 in these complexes (Fig. 1e), as detailed below.

The PGF 2α binding pocket of FP
The endogenous ligand PGF 2α is mainly composed of three parts, a carboxyl group-containing α-chain, a five-membered ring (F ring) with two hydroxyl groups, and a hydrophobic ω chain with one hydroxyl group at ω6 position (Fig. 2a). The cryo-EM map enabled the unambiguous assignment of PGF 2α within the receptor pocket. PGF 2α is well resolved in the FP ligand-binding pocket by adopting an L-shape conformation with its carboxyl group-containing α-chain fitting into a hydrophilic sub-pocket near the top of the receptor (Fig. 2a), formed by residues from TM1, TM7, and ECL2 (Fig. 2b). The carboxyl group in the α-chain of PGF 2α forms a salt bridge with R291 7.40 and hydrogen bonds with T184 EL2 and Y92 2.65 . Mutations of residues R291 7.40 , T184 EL2 , and Y92 2.65 , which are highly conserved in prostanoid receptors and participate directly in receptor binding, lead to decreased activity of PGF 2α (Fig. 2b-d, Supplementary Fig. 5-6). Although electrostatic contacts are the major driving force for the interactions between the α-chain and the positively charged binding pocket of FP, several hydrophobic residues also play important roles. Particularly, we did see the side chain of M115 3.23 forms lone pair-π interaction with ethylene linkage in the α-chain (Fig. 2b, c, Supplementary Fig. 6). Mutating this methionine in FP to alanine decreased the affinity for PGF 2α by approximately 100-fold (Fig. 2d, Supplementary Fig. 5). The F ring is located in a sub-pocket formed by TM1, TM2, and TM7 (Fig. 2b). These two hydroxyl groups in F ring mainly participate in polar interactions with the receptor. Particularly, these hydroxyl groups form hydrogen bonds with S33 1.39 and T294 7.43 , which are not conserved among prostanoid receptor family members, indicating that this part mainly contributes to the selectivity of PGF 2α to FP. Meanwhile, mutation of S33 1.39 or T294 7.43 in FP to alanine significantly impaired the affinity to PGF 2α (Fig. 2d, Supplementary Fig. 5). Besides α chain and F ring, the ω chain penetrates into the hydrophobic pocket formed by TM5, TM6, and TM7. This alkyl chain forms hydrophobic interactions with F205 5.41 , W262 6.48 , F265 6.51 , and L290 7.39 . Mutations of these residues in FP to alanine significantly impaired the affinity to PGF 2α (Fig. 2d, Supplementary Fig. 5). Besides hydrophobic interaction, the ω6 hydroxy group forms a hydrogen bond with H81 2.54 , which mutated to alanine also caused a dramatically reduced the activity of PGF 2α to FP by over 1000 folds (Fig. 2d, Supplementary Fig. 5). Collectively, the α-chain and ω-chain that bind to the sub-pockets with highly conserved residues, mainly contributes to high receptor binding affinity, while the F ring could be important for receptor selectivity.

Specific engagement of LTPA and TFPA with FP
Even though FP is an important therapeutic target for many diseases, the poor selectivity of PGF 2α has hampered its clinical application. LTPA and TFPA, two synthetic relative selective FP agonists, have been widely used in clinical treatment for diseases including glaucoma. LTPA showed high affinity to FP with an EC 50 of 3.6 nM but only moderate potency to EP1 and EP3, with an EC 50 of 6.9 and 17 μM, respectively 27 . Compared to PGF 2α , the selectivity was enhanced by more than 100 times 27 . TFPA is the most potent FP agonist (EC 50 : 0.4 nM) and has a fairly low potency to the other members of the prostanoid receptor family except for EP3 (EC 50 : 67 nM) 28 . LTPA, TFPA, and PGF 2α all have shared α-chain and F ring, while LTPA and TFPA have bulky substitutions in the ω-chain compared to PGF 2α (Fig. 3 a-e). LTPA and TFPA bond to FP in nearly identical binding poses as PGF 2α did, displaying a similar "L"-shape configuration in these two solved structures (Fig. 3a). Likewise, the carboxylate groups of LTPA and TFPA form strong polar interactions with R291 7.40 , T184 EL2 , and Y92 2.65 , the ethylene linkage in the α-chain forms a lone pair-π 29 interaction with M115 3.23 , and the two hydroxy groups in the F ring form hydrogen bonds with S33 1.39 and T294 7.43 (Fig. 3b, c). Consistent with this observation, mutations of these residues in FP to alanine significantly decreased the potency of LTPA and TFPA to FP.
The derivatized ω-chain in LTPA also directly binds to FP. The phenyl group in the ω-chain of LTPA packs against F205 5.41 , F265 6.51 , F187 ECL2 , L290 7.39 , and W262 6.48 with hydrophobic interactions. These residues are highly conserved in FP, EP1, and EP3. Interestingly, besides forming a hydrogen bond with H81 2.54 , the hydroxyl group in the ω chain forms an additional hydrogen bond with S118 3.35 , which only exists in FP ( Fig. 3d-f), indicating that this residue is of vital importance for the selectivity of LTPA to FP. Consistent with this finding, mutation of this serine to alanine or asparagine significantly reduced the potency of LTPA to induce FP activation (Fig. 3g, Supplementary Fig. 5).
Similar to LTPA, the phenyl group in the ω-chain of TFPA forms extensive hydrophobic interactions with FP. Moreover, the oxygen atom forms a hydron bond with the side chain of Q297, and the two fluoride groups in the carbon 12-position of TFPA form three hydrogen bonds with several residues in FP 30 , which may explain its high potency to FP. One fluoride group in TFPA forms hydrogen bonds to H81 2.54 and S118 3.35 , as the hydroxyl group in LTPA did. Notably, the other fluoride group forms a hydrogen bond with N84 2.57 (Fig. 3e, Supplementary  Fig. 6). This residue is highly diverse among prostanoid receptor family members. Intriguingly, among the other 8 prostanoid receptors, only EP3 and TP harbor a similar polar uncharged residue, threonine (Fig. 3f). Through structural analysis, mutation of N84 2.57 to threonine in FP could still form a hydrogen bond with the fluoride group (Supplementary Fig. 6). This explains its strong affinity for EP3. Consistent with this prediction, mutation of this residue to threonine does not significantly reduce ligand binding of TFPA, but mutating to alanine ). This hydrogen bond formed between T 2.57 in EP3 and TFPA may contribute to the high affinity of TFPA toward EP3 28 .

The active structure of FP
Structural comparison of the PGF 2α -bound FP-G q complexes with the antagonist-bound TP (PDB: 6IIU) 31 supports the notion that FP in these structures is in the active state, featured by the outward displacement of the cytoplasmic end of TM6, the hallmark of class A GPCR activation, and concurrently inward shift of TM7. In addition, TM5 of FP laterally shifts relative to that of antagonist-bound TP. These conformation changes largely resemble that of the G q -coupled 5-HT 2A R complex (PDB: 6WHA) 22 (Fig. 4a), but the outward amplitude of TM6 of FP is smaller than that of 5-HT 2A R (Fig. 4a). Structure comparison of the active FP in complex with PGF 2α and the inactive TP bound to an antagonist provide clues for understanding the activation mechanism of FP. Upon PGF 2α binding, the ωchain of PGF 2α approaches the toggle switch residue W262 6.48 to trigger the downward displacement of W262 6.48 by 2.4 Å (Fig. 4b, c). The movement of W262 6.48 further constitutes a hydrophobic LLW core triad, which is comprised of L123 3.40 , L213 5.49 , and W262 6.48 , to fasten TM3, TM5, and TM6. The importance of the LLW core triad is functionally supported by the decreased potency of PGF 2α to activate L123 3.40 A and L213 5.49 A FP mutants (Fig. 4c, Supplementary Fig. 7). Notably, the canonical P 5.50 I 3.40 F 6.44 motif in majority of class A GPCRs 26,32 is replaced by G214 5.50 L123 3.40 S258 6.44 . In our structure of active FP, G214 5.50 is sterically apart from S258 6.44 and L123 3.40 , leading to the lack of the conserved PIF hydrophobic triad, thus indicating that FP employs an activation mechanism not mediated by the traditional PIF motif (Fig. 4b).
The receptor activation also accompanies the arrangement of ionic lock (D/E 3.49 R 3.50 Y 3.51 , E132 3.49 R133 3.50 C134 3.51 in FP), leading to the broken of the salt bridge between E132 3.49 and R133 3.50 and the stretching of the R133 3.50 side chain towards TM6 and the latter's outward displacement of 6.7 Å compared with that of inactive TP (measured at Cα of residue 6.30). C134 3.51 in the ERC motif moves inwards, forming interactions with I222 5.58 and T223 5.59 in TM5. The importance of motif in FP activation is evidenced by the decreased PGF 2α activity on FP mutants of E132 3.49 , R133 3.50 , and C134 3.51 (Supplementary Fig. 7). Intriguingly, unlike its cognate residue R312 8.47 in TP, the side chain of R308 8.47 in FP undergoes a large-scale upward rotation and forms a hydrogen bond with Q250 6.36 , which leads to the inward shift of the cytoplasmic end of TM7 and the upshift of H8 (Fig. 4d) Fig. 7) Noteworthily, the arginine at position 8.47 is highly conserved across prostaglandin receptors, while Q 6.36 only exists in several class A receptors, including EP1, EP3, and FP, indicating that this interaction network could also exist in active structures of EP1 and EP3.

FP-G q coupling
The notable outward displacement of TM6 at the cytoplasmic side opens a cavity to accommodate the Gα q subunit. Structural comparisons of FP-G q with G q -coupled CCK A R and 5HT 2A R reveal a difference in the conformations of TM1, TM6, and Gα q subunits among these G qcoupled GPCR complexes (Fig. 5a). The cytoplasmic end of FP TM1 and TM6 undergoes a remarkably inward displacement relative to CCK A R and 5HT 2A R. Consequently, the C-terminus of the α5 helix of the Gα q subunit in the FP-G q complex rotates toward TM7 and H8 to avoid clashes with TM6 and forms extra hydrophobic interactions with side chains of residues in H8, accompanied by the rotation of the entire Gα q subunit (Fig. 5a). This alteration triggers a 14°tilt of the αN helix of Gα q , bringing it closer to the cytoplasmic end of TM4 in the FP-G q complex relative to CCK A R-G q and 5HT 2A R-G q complexes (Fig. 5a). In addition to coupling G q by interactions with H8 and TM4, the engagement of G q is also maintained by interactions with FP from TM1, TM2, TM3, TM5, TM4, TM6, ICL1, and ICL2. The side chain of E132 3.49 of the E 3.49 (D) R 3.50 C 3.51 (Y) motif in TM3 makes a direct hydrogen bond interaction with the side chain of the Y356 of α5 (Fig. 5b, c). Like other G q -coupled GPCR 33 , the ICL2 of FP facilitates broad interactions with G q to stabilize the complex. Typically, ICL2 adopts a helix conformation and inserts into the groove formed by the αN, β2-β3 loop, and α5 helix of Gα q . Interestingly, we identified several distinct interactions between TM6 and Gα q . For instance, the H244 6  H-bonds were depicted as red dashed lines. d, e 2D representation of contacts in LTPA-FP and TFPA-FP. The polar bonds were presented by red dotted lines. Conserved residues that formed interactions with ligands are presented in a red oval frame. f Sequence alignment of prostanoid receptors. Hydrophobic residues are in yellow, polar charged residues in blue, and polar uncharged residues in green. g, IP1 accumulation assay of key mutants in FP that bind to LTPA or TFPA (ΔpEC 50 = pEC 50 of agonists to specific Mutant FP-pEC 50 of PGF 2α to WT FP, Yellow column means ΔpEC 50 ≤ −1, Blue column means ΔpEC 50 ≤ −2). Data are presented as mean values ± SEM; n = 3 independent samples; significance was determined with two-side unpaired t-test; n.s. no significant; *p < 0.05; **p < 0.01; ***p < 0.001. Exact p values and Source data are provided as a Source Data file. and E246 6.32 forms a hydrogen bond interaction with the side chain of N357 of Gα q (Fig. 5d). Mutating of H244 6.30 and E246 6.32 to alanine destabilizes the complex and reduces the IP1 accumulation of FP (Fig. 5d, Supplementary Fig. 6). Most notably, unlike other solved G qcoupled GPCR structures, the extensive interactions of TM1, ICL1, and TM2 of FP with G q play an important role in stabilizing the complex (Fig. 5e). F58 packs against L358 and V359 of Gα q stabilized by hydrophobic interaction, and K63 packs against R37 of Gα q . S62 forms a hydrogen bond with E355 of Gα q (Fig. 5e). Mutating S62 to alanine almost abolished the IP1 accumulation of FP (Fig. 5e, Supplementary  Fig. 7), supporting that the direct interactions of TM1, ICL1, and TM2 with G protein are important for FP to couple with G protein.

Discussion
Glaucoma is the leading cause of permanent eyesight loss in the world 34 . According to the World Health Organization (WHO), 79.6 million individuals suffered from glaucoma in 2020, and the number of patients may increase to 111.8 million by 2040 35 . Activation of prostaglandin receptors like FP, EP1-EP4, and DP showed anti-glaucoma effects. Research for the discovery of pharmaceutical drugs selectively targeting each of these receptors has been extensively conducted, whereas only FP selective agonists are approved for treatment due to their minimal side-effect profile 36 . Elucidating the mechanism underlying the functional selectivity of individual FP receptors is of utmost importance for developing new drugs with higher selectivity towards individual prostaglandin receptors to avoid or reduce undesirable side effects.
In this study, we present the cryo-EM structures of FP-G q in complex with its endogenous ligand PGF 2α as well as two synthetic agonists LTPA and TFPA. Through structure analysis of the PGF 2α -FP-G q complex, the carboxylate-containing α-chain that binds to the sub-pocket with highly conserved residues contributes to the majority of the high potency of the receptor. In contrast, the F ring is important for receptor selectivity. Similar to PGE 2 to EP3, the hydrophobic ω-chain directly interacts with the toggle switch W262 6.48 in FP and forms hydrophobic interactions with a set of hydrophobic residues. The hydroxyl group in the ω-chain of LTPA forms an additional hydrogen bond with S118 3.35 in FP in the LTPA-FP-G q structure, which may be the key to LTPA's higher selectivity to FP. Despite PGF 2α containing the hydroxyl group at the same position as LTPA that the presence of the aromatic ring and lack of carbon-to-carbon double bonds in the ω-chain of LTPA may affect the geometry of the ligand, indicating that these modifications in the ω-chain of PGAs may improve their selectivity to FP. Compared with the LTPA, TFPA forms an extra hydrogen bond to N84 2.57 in FP with a fluoride group in the ω chain. EP3 harbors a similar polar uncharged residue, T 2.57 , which may form a hydrogen bond with the fluoride group of TFPA. Although TFPA had a higher affinity for FP than LTPA, its selectivity needs to be further improved. These structures revealed the ligand recognition of FP, which will serve as templates for the rational design of a new generation of potent agonists with desired selectivity profiles.
Through structural comparison and mutagenesis studies, we also elucidated the mechanisms of receptor activation and G protein coupling by FP. FP has neither the traditional 'PIF' core triad nor the DRY motif that is commonly involved in the activation of class A GPCRs, instead FP senses ligand binding by the conserved toggle switch W 6.48 to tether TM3-TM5-TM6 through a hereby identified LLW core triad consisting of L123 3.40 , L213 5.49 . Intriguingly, mutation of the W262 6.48 in FP to alanine did not entirely abolish these three agonists induced activation of FP. It is speculated that the ω chain of PGF 2α , LTPA, or TFPA in W262A mutant still induces the activation of mutated FP because it may point downward in a similar manner to EP2 (Fig. 1e), which naturally lacks W 6.48 at the toggle switch residue position 37 . In the FP receptor, the conserved D/ERY motif is replaced by ERC, which only exists in several class A GPCRs, like EP1, IP, and Neurotensin receptor type 2. Interestingly, in the active FP structure, Q250 6.36 induces an upswing of the side chain of R308 8.47 and forms a hydrogen bond with it that leads to the inward shift of the cytoplasmic end of TM7 and the upshift of H8. R 8.47 is particularly conserved in prostaglandin receptors but rarely exists in other class A GPCRs, and Q 6.36 only exists in several class A receptors, including EP1, EP3, and FP. All these key residues for activation in FP are harbored by EP1, which also primarily couples with G q . Thus, this activation mechanism should be applicable to both FP and EP1, which have the same unique residues.
The distinct features of the active FP structures also define the way of G q protein coupling. Specifically, the TM1, ICL1, and TM2 of FP form extensive interactions with G q , which, to our knowledge, haven't been shown in any other reported G q -coupled GPCR structures. These observed characteristics in the FP-G q complex structures, including the arrangement of the 7TM bundle, the ligand-binding mode, the ligand-induced receptor activation, and the manner of G protein coupling, expand the understanding of lipid recognition and GPCR-G q coupling mechanism. Collectively, our results reveal conserved and diverse mechanisms of ligand binding, receptor activation, and G protein coupling by FP. This panel is shown with views an orthogonal view (left) and a cytoplasmic view (right); the red arrows indicate the tilt of the α5 helix of Gα q from the FP-G q complex compared to the 5-HT 2A R-G q or CCK A1 -G q complexes. b the residues in FP, 5-HT 2A R, and CCK A1 that contact G q . c-e The detailed interactions of ICL2 with Gα q (c), TM6 with the α5 helix of Gα q (d) and TM1, TM2, and ICL1 with the αN and α5 helices of Gα q (e). The hydrogen bonds are depicted as dashed lines.

Constructs
The full-length human FP was modified to contain the N-terminal thermally stabilized BRIL 20 to enhance receptor expression and the addition of an N-terminal Flag tag. LgBiT was inserted at the C-terminus of the human FP using homologous recombination. The modified FP was cloned into the pFastBac (Thermo Fisher Scientific) vectors using the ClonExpress II One Step Cloning Kit (Vazyme Biotech). An engineered Gα q chimera was generated based on the mini-Gαs scaffold with its aa1-18 replaced by corresponding sequences of Gα i1 , aa348-359 replaced by corresponding sequences of Gα q , A33, H35, A87, V92, D107, V115, R137, N144, C151, F155, K158, V161, K163, D171, and D319 replaced by corresponding residues in Gα q , designated as mGα s/q/iN . Human wild-type (WT) Gβ1, human Gγ2, and a singlechain antibody scFv16 38 , as well as a Gβ1 fused with SmBiT at its Cterminus, were cloned into pFastBac vectors.

Insect cell expression
Human FP, G q chimera, Gβ1, Gγ, and scFv16 were co-expressed in High Five insect cells (Invitrogen) using the baculovirus method (Expression Systems). Cell cultures were grown in ESF 921serum-free medium (Expression Systems) to a density of 2-3 million cells per mL and then infected with six separate baculoviruses at a suitable ratio. The culture was collected by centrifugation 48 h after infection, and cell pellets were stored at −80°C.

Cryo-EM data collection
Cryo-EM grids were prepared with the Vitrobot Mark IV plunger (FEI) set to 4°C and 100% humidity. Three microliters of the sample were applied to the glow-discharged gold R1.2/1.3 holey carbon grids. The sample was incubated for 10 s on the grids before blotting for 3 s (double-sided, blot force −2) and flash-frozen in liquid ethane immediately. For FP-G q -PGF 2α complex, FP-G q -LTPA complex, and FP-G q -TFPA complex datasets, 3902, 7972, and 6513 movies were collected, respectively, on a Titan Krios equipped with a Gatan K3 direct electron detection device at 300 kV with a magnification of 105,000, corresponding to a pixel size 0.824 Å. Image acquisition was performed with EPU Software (FEI Eindhoven, Netherlands). We collected a total of 36 frames accumulating to a total dose of 50 e − Å −2 over 2.5 s exposure.

Cryo-EM image processing
MotionCor2 was used to perform the frame-based motion-correction algorithm to generate a drift-corrected micrograph for further processing, and CTFFIND4 provided the estimation of the contrast transfer function (CTF) parameters 39,40 . All subsequent steps, including particle picking and extraction, two-dimensional (2D) classification, three-dimensional (3D) classification, 3D refinement, CTF refinement, Bayesian polishing, post-processing, and local resolution estimation, were performed using Relion3.0 41 .
For FP-G q -PGF 2α complex dataset, 115 aligned micrographs were deleted because of contaminations or bad ice quality. A total of 3,391,620 particles were extracted from the cryo-EM micrographs and followed by two rounds of reference-free 2D classification, yielding 895,825 particles after clearance. Mask 3D classification on the receptor part was used to separate out 479,164 particles which resulted in a clearer density of PTGFR. We refined these particles, which led to a structure at 3.24 Å global resolution. After the postprocessing, the particles were reconstituted to a 2.67 Å structure (Supplementary Fig. 1).
For FP-G q -LTPA complex dataset, 1438 aligned micrographs were deleted because of contaminations or bad ice quality. A total of 5,478,774 particles were extracted from the cryo-EM micrographs and followed by two rounds of reference-free 2D classification, yielding 1,181,590 particles after clearance. The global 3D classification was used to separate out 437,740 particles. We then continued the processing in Relion3.0 and refined 437,740 particles, which led to a structure at 3.40 Å global resolution. After CTF refinement, Bayesian polishing, and postprocessing, then the particles were reconstituted to a 2.78 Å structure ( Supplementary Fig. 2).
For FP-G q -TFPA complex dataset, 279 aligned micrographs were deleted because of contaminations or bad ice quality. A total of 5,774,308 particles were extracted from the cryo-EM micrographs and followed by two rounds of reference-free 2D classification, yielding 2,418,674 particles after clearance. The global 3D classification was used to separate out 805,802 particles. Two rounds of mask 3D classification on the receptor part were used to separate out 578,962 particles which resulted in a clearer density of PTGFR. We refined these particles, which led to a structure at 3.35 Å global resolution. After CTF refinement, Bayesian polishing, and postprocessing, then the particles were reconstituted to a 3.14 Å structure ( Supplementary Fig. 3). We also performed postprocessing of all three final maps with DeepEMhancer 42 .

Model building
A predicted FP structure from Alphafold2 was used as the starting reference model for receptor building 43 . Structures of G αq , Gβ, Gγ, and the scFv16 were derived from PDB entry 7WKD 44 and were rigid body fit into the density. All models were fitted into the EM density map using UCSF Chimera 45 , followed by iterative rounds of manual adjustment and automated rebuilding in COOT 46 and PHENIX 47 , respectively. The model was finalized by rebuilding in ISOLDE 48 , followed by refinement in PHENIX with torsion-angle restraints to the input model. The final model statistics were validated using Comprehensive validation (cryo-EM) in PHENIX 47  Inositol phosphate accumulation assay IP-One production was measured using the IP-One HTRF kit (Cisbio) 50 . Briefly, AD293 cells (Agilent) were grown to a density of 400,000-500,000 cells per mL and then infected with separate plasmids at a suitable concentration. The culture was collected by centrifugation 24 h after incubation at 37°C in 5% CO 2 with a Stimulation Buffer. The cell suspension was then dispensed in a white 384well plate at a volume of 7 μl per well before adding 7 μl of ligands. The mixture was incubated for 1 h at 37°C. IP-One-d2 and anti-IP-One Cryptate dissolved in Lysis Buffer (3 μl each) were subsequently added and incubated for 15-30 min at room temperature before measurement. Intracellular IP-One measurement was carried out with the IP-One HTRF kit and EnVision multi-plate reader (PerkinElmer) according to the manufacturer's instructions. Data were normalized to the baseline response of the ligand. pEC 50 E min , and E max for each curve were calculated by GraphPad Prism 8.0. ΔpEC 50 equals pEC 50 of agonists to specific Mutant minus pEC 50 of agonists to WT. Data are presented as mean values ± SEM; n = 3 independent samples; n.s. no significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Receptor surface expression
Cell-surface expression levels of WT or mutants FP were quantified by flow cytometry. AD293 cells were seeded at a density of 1.5 × 10 5 per well into 12-well culture plates. Cells were grown overnight and then transfected with 1.0 μg FP construct by FuGENE® HD transfection reagent in each well for 24 h. After 24 h of transfection, cells were washed once with PBS and then detached with 0.2% (w/v) EDTA in PBS. Cells were blocked with PBS containing 5% (w/v) BSA for 15 min at room temperature before incubating with primary anti-Flag antibody (diluted with PBS containing 5% BSA at a ratio of 1:300, Sigma-Aldrich, F3165) for 1 h at room temperature. Cells were then washed three times with PBS containing 1% (w/v) BSA and then incubated with anti-mouse Alexa-488conjugated secondary antibody (diluted at a ratio of 1:1000, Thermo Fisher, A-11029) at 4°C in the dark for 1 h. After another three times of washing, cells were collected, and fluorescence intensity was quantified in a Luminex flow cytometer system (Guava® easyCyte) through a Luminex guavaSoft 4.5 at excitation 488 nm and emission 519 nm. Approximately 10,000 cellular events per sample were collected, and data were normalized to the wild-type FP. Experiments were performed at least three times, and data were presented as means ± SEM.

Statistics
All functional study data were analyzed using GraphPad Prism 8.0 (Graphpad Software Inc.) and showed as means ± S.E.M. from at least three independent experiments in triplicate. The significance was determined with a two-sided, unpaired t-test, and *p < 0.05 was considered statistically significant.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The atomic coordinates and the electron microscopy maps have been deposited in the Protein Data Bank (PDB) under accession numbers 8IUK, 8IUL, and 8IUM and Electron Microscopy Data Bank (EMDB) accession number EMD-35724, EMD-35725, and EMD-35726 for the PGF2α-FP-G q and the LTPA-FP-G q and the TFPA-FP-G q complex, respectively. Previously published structures can be accessed via accession codes: 7CX2; 7D7M; 6IIU; 6WHY; 7MBY; 6WHA; 7WKD. Source data are provided in this paper.
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